The robustness of porin-cytochrome gene clusters from Geobacter metallireducens in extracellular electron transfer

ABSTRACT To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome (pcc) gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens. Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens. Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens ΔhybLΔfdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910. Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET. IMPORTANCE The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome (pcc) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens. In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens. The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.

form coculture with M. barkeri, M. acetivorans, or M. subterranea during the initial stage of coculture.These mutants then regained their ability to form cocultures with these archaea in the late stage of coculturing.Deletions of Gmet0910 or Gmet0913 also lowered the bacterial ability to form coculture with G. sulfurreducens (10).Further investigation demonstrated the increased mRNA abundance of Gmet0908-0910 when cocultured with M. thermoacetophila, suggesting their importance in DIET from G. metallireducens to M. thermoacetophila (11).
Despite above advances, the roles of Gmet0825-0828 in EET have never been experimentally investigated.Moreover, the contributing roles of these complexes in EET have never been systematically compared.To these ends, the gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 were each deleted and a double mutant without Gmet0825-0828 and Gmet0908-0910 (ΔGmet0825-0828ΔGmet0908-0910) was also constructed.Previous results showed the deletion or modification of the pilA-N gene for G. metallireducens impaired bacterial EET ability (8,29,30).Thus, the pilA-N gene of G. metallireducens was also deleted and ΔpilA-N served as a control.The resulting mutants were tested for their ability to reduce Fe(III)-citrate, ferrihydrite, and anodes as well as to form cocultures with M. barkeri and the wild type (WT) and ΔhybLΔfdnG of G. sulfurreducens.Results showed that the three pcc gene clusters of G. metalliredu cens could not be deleted at the same time, suggesting their unknown essential roles or the regulatory effects of these clusters in anaerobic growth of G. metallireducens unrelated to EET.The gene-cluster-deletion mutants also displayed varied abilities to reduce Fe(III)-citrate, ferrihydrite, and anodes and to form cocultures with M. barkeri and G. sulfurreducens, demonstrating that the Pcc complexes contributed to the EET of G. metallireducens differently.The involvement of Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 in ferrihydrite reduction further suggested their overlapping roles in ferrihydrite reduction.Most importantly, the findings that the mutant without either Gmet0825-0828 or Gmet0908-0910 regained its EET ability and that deletion of these clusters increased expression of Gmet0911-0913 provided new evidence for regulatory complexity in these pcc gene clusters.
The constructed mutants were compared for their ability to reduce Fe(III)-citrate and ferrihydrite with that of the WT of G. metallireducens.Except for ΔGmet0911-0913, all other mutants showed the similar ability of reducing Fe(III)-citrate to that of WT.ΔGmet0911-0913 displayed delayed ability to reduce Fe(III)-citrate, as compared with that of WT and other mutants of G. metallireducens (Fig. 2A).Compared with that with empty vector, the ability of ΔGmet0911-0913 complemented with cloned Gmet0911-0913 for Fe(III)-citrate reduction was significantly increased (Fig. 2B).
Except for ΔGmet0825-0828ΔGmet0908-0910, all other mutants showed diminished ability to reduce ferrihydrite, as compared with that of the WT of G. metallireducens (Fig. 2C).At 10 days after inoculation, reductions of ferrihydrite by WT and ΔGmet0825-0828ΔGmet0908-0910 plateaued, while other mutants reduced ferrihydrite at much slower rates.After that, ΔGmet0825-0828, ΔGmet0908-0910, ΔGmet0911-0913, and ΔpilA-N reduced ferrihydrite at different rates.At 26 days after inoculations, the ability to reduce ferrihydrite decreased in the order of ΔGmet0908-0910 > ΔGmet0911-0913 > ΔpilA-N > ΔGmet0825-0828 (Fig. 2C).Compared with those with empty vector, all singlegene-cluster-deletion mutants complemented with their respective gene cluster showed some increased ability to reduce ferrihydrite (between 33% and 63% of WT) but in no case was complementation able to recover WT reduction (Fig. 2D).

Anode reduction
The electricity production by the WT of G. metallireducens was detected at 1 day after growth and plateaued at 2 days after growth (Fig. 3A).Compared with the WT, ΔGmet0825-0828ΔGmet0908-0910 showed a delayed ability to produce electricity on anodes.The electricity production of ΔGmet0825-0828ΔGmet0908-0910 plateaued at 3 days after growth (Fig. 3A).Little electricity was produced by ΔGmet0911-0913 during the first 3 days of growth.The electricity production by ΔGmet0911-0913 then increased (Fig. 3A).During 8 days of growth, little electricity was produced by ΔGmet0825-0828, ΔGmet0908-0910, or ΔpilA-N (Fig. 3A).The maximum electricity production detected decreased in the order of WT ≈ ΔGmet0825-0828ΔGmet0908-0910 >> ΔGmet0911-0913 >> ΔGmet0825-0828 ≈ ΔGmet0908-0910 ≈ ΔpilA-N (Fig. 3B).The amounts of protein and biofilm detected on the electrode surface also decreased in the same order (Fig. 3C  through I).

Coculture with M. barkeri
In the first generation, all other mutants could coculture with M. barkeri, except for ΔGmet0825-0828 that could not form coculture with M. barkeri (Fig. 4A  through D).Compared with that of the WT of G. metallireducens, the coculture of ΔGmet0825-0828ΔGmet0908-0910 oxidized ethanol more quickly (Fig. 4A and B).However, no major difference was detected in methane production (Fig. 4C) and maximum copies of microbial 16S rRNA genes (Fig. 4D) between the coculture with the WT and that of ΔGmet0825-0828ΔGmet0908-0910.Compared with that of the WT and ΔGmet0825-0828ΔGmet0908-0910, the cocultures of ΔGmet0908-0910, ΔGmet0911-0913, or ΔpilA-N showed delayed ability to oxidize ethanol and to produce methane (Fig. 4A  through C).The maximum copies of microbial 16S rRNA genes detected in the cocultures of ΔGmet0911-0913 and ΔpilA-N were similar to each other, but lower than that of WT and ΔGmet0825-0828ΔGmet0908-0910 and higher than that of ΔGmet0908-0910 (Fig. 4D; Table S4).
In the second generation, the cocultures with WT and ΔGmet0825-0828ΔGmet0908-0910 displayed no difference in terms of their oxidation of ethanol and formation of methane (Fig. S1A through C).However, the maximum copies of 16S rRNA genes detected in the coculture with the WT was lower than that with ΔGmet0825-0828ΔGmet0908-0910 (Fig. S1D).The ability of the coculture with ΔGmet0911-0913 to oxidize ethanol and to produce methane was lower than that with WT and ΔGmet0825-0828ΔGmet0908-0910 but higher than that with ΔGmet0908-0910 and ΔpilA-N (Fig. S1A through C).Similarly, the maximum copies of microbial 16S RNA genes detected in the cocultures of ΔGmet0911-0913 were also lower than those of WT and ΔGmet0825-0828ΔGmet0908-0910, but higher than those of ΔGmet0908-0910 and ΔpilA-N (Fig. S1D; Table S4).Fluorescence in situ hybridization (FISH) analyses of the formed granules confirmed the coculture of G. metallireducens-M.barkeri (Fig. S2A and B).
In the second generation of cocultures, ΔpilA-N and ΔGmet0825-0828 of G. met allireducens could not grow with the WT of G. sulfurreducens.The cocultures with the WT, ΔGmet0911-0913, and ΔGmet0825-0828ΔGmet0908-0910 were similar in terms of their ethanol oxidation, fumarate reduction, malate metabolism, and maximum copies of bacterial 16S rRNA genes detected (Fig. S3).Compared with that with the WT, ΔGmet0911-0913, and ΔGmet0825-0828ΔGmet0908-0910, the coculture with ΔGmet0908-0910 showed a reduced ability to oxidize ethanol and to reduce fumarate (Fig. S3A through D).Similarly, maximum copies of bacterial 16S rRNA genes detected in the coculture with ΔGmet0908-0910 were lower than those in the cocultures with the WT, ΔGmet0911-0913, andΔGmet0825-0828ΔGmet0908-0910 (Fig. S3E; Table S5).The coculture of G. metallireducens-G.sulfurreducens was also confirmed by FISH analyses (Fig. S2C and D).
Previous results indicated that indirect interspecies electron transfer from G. metallireducens to G. sulfurreducens, such as that mediated by H 2 , could overshadow the roles of bacterial genes in DIET from G. metallireducens to G. sulfurreducens (31).To avoid interference from indirect interspecies electron transfer, the hybL and fdnG genes for G. sulfurreducens were deleted, as they were involved in indirect interspecies electron transfer (32).The WT and mutants of G. metallireducens were then cocultured with G. sulfurreducens ΔhybLΔfdnG.Compared with that with the WT of G. metallireducens and WT of G. sulfurreducens, the coculture with the WT of G. metallireducens and G. sulfurreducens ΔhybLΔfdnG was enhanced (Fig. S4 and S5).Similar to that with the WT of G. sulfurreducens, the mutants of G. metallireducens could coculture with G. sulfurreducens ΔhybLΔfdnG in the first generation and their ability to form coculture was lower than that with the WT of G. metallireducens (Fig. S4; Table S6).In the second generation of cocultures, WT, ΔGmet0911-0913, and ΔGmet0825-0828ΔGmet0908-0910 of G. metallire ducens grew well with G. sulfurreducens ΔhybLΔfdnG; ΔGmet0908-0910 grew poorly with G. sulfurreducens ΔhybLΔfdnG; and ΔpilA-N and ΔGmet0825-0828 could not grow with G. sulfurreducens ΔhybLΔfdnG (Fig. S5; Table S6).These were similar to those grown with the WT of G. sulfurreducens except that ΔGmet0908-0910 grew more poorly with G. sulfurreducens ΔhybLΔfdnG than with WT of G. sulfurreducens (Fig. S3; Table S5) sulfurreducens ΔhybLΔfdnG (Fig. S6).Compared with that of the coculture with the WT of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG, the mRNA levels of 808 genes of G. metallireducens were elevated and 589 genes of G. metallireducens were decreased in the coculture with ΔGmet0825-0828 of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG (Fig. S7A and C; Table S7).These included eight upregulated c-Cyt genes and 29 downregu lated c-Cyt genes (Table S7).The mRNA levels of 93 genes of G. metallireducens were elevated and 146 genes of G. metallireducens were decreased in the coculture with ΔGmet0825-0828ΔGmet0908-0910 of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG, as compared with that in the coculture with the WT of G. metallireducens-G.sulfurre ducens ΔhybLLΔfdnG (Fig. S7B and C; Table S8).These included 13 upregulated c-Cyt genes and 15 downregulated c-Cyt genes (Table S8).Notably, Gmet0911-0913 were among the genes whose mRNA levels increased 1.47-to 1.65-fold in the coculture with ΔGmet0825-0828ΔGmet0908-0910 of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG, as compared with that with the WT of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG (Fig. 6A; Table S9).The mRNA levels of Gmet0911-0913 in the coculture with ΔGmet0825-0828 of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG, however, were 25%-42% of that in the coculture with the WT of G. metallireducens-G.sulfurreducens ΔhybLΔfdnG (Fig. 6A; Table S9).The mRNA levels of housekeeping gene dnaK of G. metallireducens remained constant in all the cocultures tested (Fig. 6A; Table S9).

Transcriptomic and immunoblot analyses
To verify the transcriptomic results above, the polyclonal antibodies specific for Gmet0913 or DnaK were generated (Fig. S8).The protein levels of Gmet0913 or DnaK were compared with the antibodies in WT, ΔGmet0825-0828, ΔGmet0908-0910, and ΔGmet0825-0828ΔGmet0908-0910 of G. metallireducens at 24 hours after reduction of Fe(III)-citrate, 10 days after reduction of ferrihydrite and 14 days after the first generation of their cocultures with G. sulfurreducens ΔhybLΔfdnG.The results consis tently showed that the protein levels of Gmet0913 in ΔGmet0825-0828ΔGmet0908-0910 appeared higher than those in the WT, ΔGmet0825-0828, and ΔGmet0908-0910 under the conditions tested.The levels of Gmet0913 in the WT appeared to be slightly higher than those in ΔGmet0825-0828 but appeared similar to that in ΔGmet0908-0910.The protein levels of DnaK were constant in all strains tested (Fig. 6B).

DISCUSSION
Previous results showed that deletion or modification of pilA-N of G. metallireducens did not impact the bacterial ability to reduce Fe(III)-citrate but diminished the bacterial ability to reduce Fe(III)-oxides and anodes and to form cocultures (8,29,30).Deletion of Gmet0910, Gmet0912, or Gmet0913 negatively impacted the bacterial ability to form cocultures (10).Our results were consistent with these previous results.
Previous results also showed that deletion of Gmet0910, Gmet0912, or Gmet0913 of G. metallireducens had little impact on the bacterial ability to reduce Fe(III)-citrate and Fe(III)-oxides (28).Our results, however, showed that deletion of Gmet0908-0910 decreased the bacterial ability to reduce ferrihydrite, while deletion of Gmet0911-0913 diminished the bacterial ability to reduce Fe(III)-citrate and ferrihydrite.This apparent discrepancy between our current and previous results is probably attributed to different bacterial mutants used in these studies.Previous investigation used single-gene-deletion mutants (28), while this investigation used single-gene-cluster-deletion mutants.Our results also showed that deletion of Gmet0825-0828 had no impact on the bacterial ability to reduce Fe(III)-citrate but substantially decreased the bacterial ability to reduce ferrihydrite.Compared with that with empty vector, complementation of the singlegene-cluster-deletion mutants with their respective gene clusters fully improved the ability of the mutant to reduce Fe(III)-citrate but only partially improved the ability of the mutants to reduce ferrihydrite.The reasons for the partial complementation observed in this investigation are currently unknown.
In solution, multiple species of Fe(III)-citrate exist (33,34).Their molecular masses are larger than 600 daltons that is the molecular mass cut-off for water-soluble mole cules to pass through the outer membrane freely (33)(34)(35).Thus, bacterial reduction of Fe(III)-citrate occurred extracellularly.Involvement of Gmet0911-0913 in extracellu lar reduction of Fe(III)-citrate not only was consistent with previous results showing the crucial roles of different protein porin-cytochrome complexes in the extracellular reduction of Fe(III)-citrate (17,25,26,36) but also might contribute our failure of constructing the mutant without Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 as the procedure for selecting the mutant was conducted under a Fe(III)-citrate-respiring condition.Deletion of Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 probably rendered the mutant unable to grow via extracellular respiration of Fe(III)-citrate, which suggests the essential role of these pcc gene clusters in the EET of G. metallireducens.Furthermore, our result showed for the first time involvements of Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 in the extracellular reduction of anodes and involvement of Gmet0825-0828 in the formation of cocultures between G. metallireducens and M. barkeri or the WT or G. sulfurreducens ΔhybLΔfdnG.Involvement of these pcc gene clusters in extracellular reduction of ferrihydrite demonstrates their overlapping roles in ferrihydrite reduction by G. metallireducens.The overlapping EET functions of these pcc gene cluster could help bacterial survival when one of the pcc gene clusters inactivated by mutations.
Although they were involved in the extracellular reduction of Fe(III)-citrate, ferrihy drite, and/or anodes and coculture formation, the contributing roles of Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 to these EET reactions varied substantially.Compared with that of Gmet0908-0910 and Gmet0911-0913, deletion of Gmet0825-0828 impacted more negatively on the extracellular reduction of ferrihydrite and cocultures between G. metallireducens and M. barkeri or the WT or G. sulfurreducens ΔhybLΔfdnG.It should be noted that deletion of Gmet0825-0828 impacted expressions of many other genes.Thus, its negative impact on extracellular reduction of ferrihydrite and cocultures between G. metallireducens and M. barkeri or the WT or G. sulfurreducens ΔhybLΔfdnG might be attributed to the direct loss of this pcc gene cluster and/or altered expression of other genes.Moreover, failure of ΔGmet0825-0828 to reduce anodes and to form any stable coculture with either M. barkeri or the WT or G. sulfurreducens ΔhybLΔfdnG clearly demonstrated the indispensable roles of Gmet0825-0828 in extracellular reduction of anodes and DIET from G. metallireducens to M. barkeri as well as the WT and ΔhybLΔfdnG of G. sulfurreducens.It should be noted that the mRNA levels of Gmet0911-0913 and the protein level of Gmet0913 were also lower than those in the WT, which might contribute to the phenotypes of ΔGmet0825-0828 in the EET observed in this study.Similarly, Gmet0908-0910 is also indispensable for extracellular reduction of anodes.Compared with Gmet0911-0913, Gmet0908-0910 played no apparent role in Fe(III)-citrate reduction, a less dominant role in ferrihydrite reduction, but more dominant roles in DIET from G. metallireducens to M. barkeri and the WT and G. sulfurreducens ΔhybLΔfdnG.Gmet0911-0913 was involved in reductions of Fe(III)-citrate, ferrihydrite, and anodes, but its roles in the cocultures were trivial.Collectively, these results clearly showed the distinct roles of these pcc gene clusters in the different EET reactions of G. metalliredu cens.The distinct EET functions of its pcc gene clusters enable G. metallireducens to use different electron acceptors more efficiently during EET reactions.
Most importantly, our results also showed for the first time that the ability of the double-gene-cluster-deletion-mutantΔGmet0825-0828ΔGmet0908-0910 to reduce ferrihydrite and anodes and to mediate DIET from G. metallireducens to M. bar keri and the WT and G. sulfurreducens ΔhybLΔfdnG increased significantly, as com pared with that of the single-gene-cluster-deletion mutants of ΔGmet0825-0828 and ΔGmet0908-0910.Our results further revealed that the improved EET capabilities of ΔGmet0825-0828ΔGmet0908-0910 were attributed at least in part to the elevated mRNA levels of Gmet0911-0913 and protein levels of Gmet0913 and probably Gmet0911 and Gmet0912.Thus, G. metallireducens might compensate for the loss of EET functions of Gmet0825-0828 and Gmet0908-0910 via at least in part the increased expression of Gmet0911-0913.The compensatory function of Gmet0911-0913 in the EET of G. metallireducens is hypothesized to have substantially improved bacterial growth when both Gmet0825-0828 and Gmet0908-0910 were deleted.This discovered compensatory function differs significantly from the improved anode reduction of the mutant from G. sulfurreducens with only the extABCD pcc gene cluster (extABCD + ) (24,25).The improved EET capability of extABCD + was only observed in anode reduction via the mechanisms other than increased expression of extABCD, such as improved biofilm formation on anodes and metabolic activity (24,25).Additionally, ΔGmet0825-0828, ΔGmet0908-0910, and ΔGmet0911-0913 behaved differently from their counterparts in G. sulfurreducens in terms of Fe(III) and anode reductions.For example, ΔGmet0911-0913 showed dimin ished reduction of Fe(III)-citrate, while ΔomaBombBomcB and ΔomaCombComcC of G. sulfurreducens reduced Fe(III)-citrate normally (17,25).These results indicated that the pcc homologs in Geobacter spp.might function differently.
Collectively, the results of this investigation demonstrated not only the essential but also the distinct, overlapping as well as compensatory roles of the pcc gene clusters in the EET of G. metallireducens.The distinct EET functions of the pcc gene clusters permit G. metallireducens to transfer electrons efficiently to the extracellular acceptors of different properties.The overlapping and compensatory EET functions enable G. metallireducens to mediate EET for bacterial growth even after two of its three gene clusters become defective by mutations.These results provide new insights into the robust roles of pcc gene clusters in bacterial EET.

Construction of mutants for G. metallireducens and G. sulfurreducens
The mutants without pcc gene clusters or pilA-N gene for G. metallireducens or without hybL and fdnG genes for G. sulfurreducens were constructed by following previously published protocols (17,26,29,31,(38)(39)(40). Briefly, three fragments were generated through PCR amplification for the single gene deletion: 500 bp upstream fragment and 500 bp downstream fragment of the target gene and the spectinomycin resist ance cassette flanked by the loxP site.These fragments and linearized plasmid pUC19 were joined with the In-Fusion HD Cloning Kit (Takara Biomedical Technology, Beijing, China) to generate a circle plasmid.After verification with DNA sequencing of inserts, the plasmids were cut with ScaI (NEB, Ipswich, MA, USA) and electroporated into the electrocompetent Geobacter cells.PCR and DNA sequencing of the deleted regions were used for verifying the constructed mutants.After verification, the spectinomycin resistance cassette was excised from the chromosome by expressing the Cre recombi nase from plasmid pCM158 (29).Double gene deletion mutants were made by repeating the above steps.The verified mutants of G. metallireducens were complemented with their respective genes cloned into the plasmid pCM66 separately (41).Escherichia coli DH5α was used for cloning and purchased from Takara Biomedical Technology (Beijing, China) (42) (Table S1).The mutants of G. metallireducens were also transformed with empty vector pCM66 as controls.Kanamycin was used at a final concentration of 200 µg/mL in the complement assays.Tables S1 and S2 list all microbial strains and plasmids and oligonucleotide primers used in this investigation, respectively.

Reductions of Fe(III)-citrate and ferrihydrite
The constructed mutants of G. metallireducens were tested for their ability to reduce Fe(III)-citrate and ferrihydrite.Ferrihydrite was prepared and characterized by following a protocol described previously (31,36,43).Briefly, ferrihydrite was synthesized by dropwise addition of 1 M NaOH to 500 mL of 0.2 M FeCl 3 until pH 7.0 was reached.The suspension was centrifuged (30 min, 2,100 × g, 20°C), washed with doubly deion ized water (ddH 2 O) prepared with a Milli-Q system (Millipore, Billerica, MA, USA), and freeze dried.X-ray diffraction with a Bruker AXS GmbH D8-Focus-Power Diffraction System (Bruker, Billerica, MA, USA), scanning electron microscopy with a HITACHI SU8010 microscope (Hitachi, Chiyoda, Japan), transmission electron microscopy with a Tecnai G20 TWIN microscope (Thermo Fisher Scientific, Waltham, MA, USA), and an ASAP 2460 Accelerated Surface Area & Porosity Analyzer (Micromeritics, Norcross, GA, USA) were used to characterize the synthesized ferrihydrite.The characterized ferrihydrite was stored at 4°C and used within 60 days.Reductions of Fe(III)-citrate and ferrihydrite were measured as described before (17,26,31).

Growth on anodes
Growth of the WT and mutants for G. metallireducens on anodes (polished graphite plate of 3.2 cm 2 ) as the sole electron acceptor was carried in microbial fuel cells (MFCs) of a single chamber with three electrodes (44).The current production of MFCs was monitored with a potentiostat (+300 mV versus Ag/AgCl) (CH Instruments Inc., Shanghai, China).At the end of each growth, the total amount of the proteins on the anode was measured with the Qubit Protein Assay Kit (Invitrogen Life Technologies, Carlsbad, CA, USA) and a Qubit Fluorometer.The biofilms formed on the anodes were visualized with a Leica TCS SP8 MP Multiphoton Confocal Microscope (Wetzlar, Germany) after being stained with the LIVE/DEAD BacLight Bacterial Viability Kits (Thermo Fisher Scientific China Co. Ltd., Shanghai, China).

Cocultures
Prior to coculture, the WT and mutants of G. metallireducens were grown in the bicarbonate-buffered medium of 56 mM Fe(III)-citrate in which acetate was replaced with 20 mM ethanol (7,8).The preadapted strains of G. metallireducens were then cocultured with M. barkeri or the WT or ΔhybLΔfdnG of G. sulfurreducens.The cocultures between the strains of G. metallireducens and M. barkeri were conducted at 37°C in the modified DSM 120 medium in which acetate was replaced with 20 mM ethanol (8), while the cocultures between the strains of G. metallireducens and the strains of G. sulfurreducens were conducted at 30°C in the bicarbonate-buffered medium of 20 mM ethanol and 40 mM fumarate (7).
During the cocultures, the concentrations of ethanol and organic acids were measured with a LC-20A Shimadzu high-performance liquid chromatography (HPLC) system that also contained a SPD-M20A UV detector, a RID-20A high-sensitivity refractive index detector (Shimadzu, Kyoto, Japan), and an Aminex HPX-87H Ion Exclusion column (300 × 7.8 mm) (Bio-Rad Laboratories Inc., California, USA).The ethanol and organic acids were purchased from Sinopharm Chemical Reagent Co. Ltd.The methane produced during the cocultures between the strains of G. metallireducens and M. barkeri was measured with a GC-2014 Shimadzu gas chromatograph system with as a flame ionization detector.Methane (99.99% purity) was purchased from Wuhan Steel Group Commercial Gasses Co. Ltd. (Wuhan, China).The copies of microbial 16S rRNA genes were measured with a QuantStudio3 (Thermo Fisher Scientific China Co. Ltd.) via quantitative PCR (qPCR) with TB Green Premix Ex Taq II (Takara Biomedical Technol ogy, Beijing, China).The microbial genomic DNAs of cocultures were isolated with the TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China).The granules formed after cocultures were examined with a Leica TCS SP8 MP multiphoton confocal microscope after FISH.The primers used in qPCR and FISH are listed in Table S2.

RNA extraction and transcriptomic analysis
Microbial cells of cocultures between the strains of G. metallireducens and G. sulfurre ducens ΔhybLΔfdnG were harvested at 4°C by centrifugation (5,000 × g, 15 min) at a predetermined time point and then frozen with liquid nitrogen.RNA was extracted using a Magen HiPure Bacterial RNA Kit (Magen Biotechnology Co. Ltd., Guangdong, China) and verified on an agarose gel.rRNA was removed with the Epicentre Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA).The cDNA libraries were constructed using a NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) and then sequenced using an Illumina NovaSeq PE150 platform at the Guangdong Magigene Biotechnology Co. Ltd., China (Guangdong, China).
Trimmomatic was used to trim the raw reads (45).Bowtie2 combined with RefSeq and Rfam 14 was used to further remove rRNA reads from trimmed reads (46)(47)(48).The non-rRNA reads were compared with the genomes of G. metallireducens (NC_007517.1) and G. sulfurreducens (NC_002939.5) with Bowtie2.The transcripts per million (TPM) method was used to normalize the gene abundances in their respective genomes (49).
The edgeR was used to analyze differential expressions of genes (50).RStudio was used to visualize the results of principal component analysis of Bray-Curtis distance and MD plots methods.

Antibody productions and immunoblot analyses
The Gme0913-or DnaK-specific antibodies were made by Proteintech Group, Inc. (Wuhan, China), and were characterized by following the protocols described before (14,51).The polypeptides used for making these polyclonal antibodies are listed in Table S3.The cells of WT and mutants of G. metallireducens that were in reductions of Fe(III)-citrate or ferrihydrite or cocultured with G. sulfurreducens ΔhybLΔfdnG were harvested at 4°C by centrifugation (5,000 × g, 15 min) at predetermined time points.The cells were washed with ice-cold Tris-buffered saline (TBS; pH 7.6) three times and were then re-suspended in TBS to the same optical density at 600 nm (OD 600 ).Equal-amount cells were lysed with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)loading buffer.The cell lysates were loaded on 10% SDS-polyacrylamide gels and then separa ted by SDS-PAGE.The separated proteins were transferred to polyvinylidene flouride (PVDF) membranes (Thermo Fisher Scientific China Co. Ltd.).The membranes were then analyzed with Gme0913-or DnaK-specific antibodies.Goat anti-rabbit IgG-HRP and pageruler prestained protein ladder were purchased from TransGen Biotech (Beijing, China) and Thermo Fisher Scientific China Co. Ltd,.respectively.The interactions between antibodies and their respective target proteins were visualized with the super sensitive ECL luminescence reagent (Meilunbio, Dalian, China) and detected with Azure C300 (Azure Biosystems Inc, Dublin, CA, USA).

Statistical analyses
All values are expressed as means ± standard deviations.Student's t test was used for comparing groups.

FIG 3
FIG 3 Anode reduction by Geobacter metallireducens and its mutants.(A) Current production.(B) Maximum current generated.(C) Bacterial protein content on anodes.All results are reported as mean and standard error of the mean (n = 3).For points with no error bar, the error was smaller than the size of the symbol.(D-I) Biofilms on anodes of different bacterial strains.In panels B and C, Student's t test was used for comparing WT and the mutants.ns, P > 0.05 and ***P ≤ 0.001.WT, wild type of G. metallireducens; Δ1, ΔGmet0825-0828; Δ2, ΔGmet0908-0910; Δ3, ΔGmet0911-0913; Δ4, ΔGmet0825-0828ΔGmet0908-0910; Δ5, ΔpilA-N.

FIG 5
FIG 5 The first generation of cocultures between WT and gene-deletion mutants of Gme and Geobacter sulfurreducens (Gsu).(A) Ethanol metabolism.(B) Fumarate metabolism.(C) Malate metabolism.(D) Succinate production.(E) The copies of combined bacterial 16S rRNA genes.All results are reported as mean and standard error of the mean (n = 3).For points with no error bar, the error was smaller than the size of the symbol.In panel E, samples were collected (Continued on next page)